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. 2013 Aug 1;3(1):19.
doi: 10.1186/2044-5040-3-19.

Measuring microRNA Reporter Activity in Skeletal Muscle Using Hydrodynamic Limb Vein Injection of Plasmid DNA Combined With in Vivo Imaging

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Free PMC article

Measuring microRNA Reporter Activity in Skeletal Muscle Using Hydrodynamic Limb Vein Injection of Plasmid DNA Combined With in Vivo Imaging

Martin G Guess et al. Skelet Muscle. .
Free PMC article

Abstract

Background: microRNA regulation plays an important role in the remodeling that occurs in response to pathologic and physiologic stimuli in skeletal muscle. In response to stress, microRNAs are dynamically regulated, resulting in a widespread "fine-tuning" of gene expression. An understanding of this dynamic regulation is critical to targeting future therapeutic strategies. Experiments elucidating this dynamic regulation have typically relied on in vitro reporter assays, ex vivo sample analysis, and transgenic mouse studies. Surprisingly, no experimental method to date allows rapid in vivo analysis of microRNA activity in mammals.

Methods: To improve microRNA studies we have developed a novel reporter assay for the measurement of skeletal muscle microRNA activity in vivo. To minimize muscle damage, hydrodynamic limb vein injection was used for the introduction of plasmid DNA encoding bioluminescent and fluorescent reporters, including click-beetle luciferase and the far-red fluorescent protein mKATE. We then applied this technique to the measurement of miR-206 activity in dystrophic mdx4cv animals.

Results: We found that hydrodynamic limb vein injection is minimally damaging to myofibers, and as a result no induction of muscle-specific miR-206 (indicative of an injury response) was detected. Unlike intramuscular injection or electroporation, we found that hydrodynamic limb vein injection results in dispersed reporter expression across multiple hindlimb muscle groups. Additionally, by utilizing click-beetle luciferase from Pyrophorus plagiophthalamus as a reporter and the far-red fluorescent protein mKATE for normalization, we show as a proof of principle that we can detect elevated miR-206 activity in mdx4cv animals when compared to C57Bl/6 controls.

Conclusion: Hydrodynamic limb vein injection of plasmid DNA followed by in vivo bioluminescent imaging is a novel assay for the detection of reporter activity in skeletal muscle in vivo. We believe that this method will allow for the rapid and precise detection of both transcriptional and post-transcriptional regulation of gene expression in response to skeletal muscle stress. Additionally, given the post-mitotic status of myofibers and stable expression of plasmid DNA, we believe this method will reduce biological variability in animal studies by allowing longitudinal studies of the same animal cohort.

Figures

Figure 1
Figure 1
miR-206 levels after hydrodynamic limb vein injection. (A) Schematic depiction of hydrodynamic limb vein injection (HLV) of plasmid DNA followed by in vivo bioluminescent and fluorescent imaging (BLI). (B) miR-206 expression in 3 to 4 month-old C57Bl/6 mice receiving either HLV injection of saline solution or BaCl2 injury, sacrificed at the indicated time points, normalized to sno202. Relative levels measured in the right (treated) gastrocnemius are displayed as mean values normalized to contralateral controls; n= 3 or 4 animals/group, Error bars = standard error of the mean. *P ≤0.05, **P ≤0.001.
Figure 2
Figure 2
Reporter distribution. Reporter expression after hydrodynamic limb vein injection. Left column: green fluorescent protein (GFP) epifluorescent signal displayed as inverted grayscale image, percentages are an average of GFP-positive fibers for indicated muscles. Right column: immunostaining for laminin (red) and epifluorescence for GFP (green) and 4′,6-diamidino-2-phenylindole (DAPI) (blue) to show distribution of GFP-positive myofibers in indicated muscles; n=1 animal. Scale bars = 100 μm. TA, tibialis anterior.
Figure 3
Figure 3
Reporter detection using in vivo bioluminescent imaging. Mice were injected with pcDNA-mKATE (top) or pcDNA-mKATE and pCBG99-Luc-Control (bottom). Data collected using Cy5.5 excitation/emission filters is shown in the left column, and after D-luciferin injection in the right column.
Figure 4
Figure 4
Quantification of miR-206 reporter activity in mdx4cv mice. (A) miR-206 expression measured in the gastrocnemius muscles of 3 month-old mdx4cv animals in comparison to C57Bl/6. miR-206 expression is normalized to sno202 ( n = 4 animals/group), mean values are displayed; error bars = standard error of the mean (SEM), *P ≤0.05. (B) Bioluminescent imaging of mdx4cv animals showing CBG99 luciferase and mKATE expression. Left hindlimbs were co-injected with pCBG99-2x-miR-206 and pcDNA-mKATE, and right hindlimbs were co-injected with pCBG99-Control and pcDNA-mKATE. (C) Region of interest (ROI) analysis of (B). The same region size was used for all animals (n= 4 animals/group; mean values are displayed; error bars = SEM, *P≤0.05.
Figure 5
Figure 5
Reporter expression in regenerating mdx4cv fibers. pCMV-eGFP-injected mdx4cv (left column) and C57Bl/6 (right column) gastrocnemius sections showing desmin immunostaining (red), green fluorescent protein (GFP) epifluorescence (green), and 4′,6-diamidino-2-phenylindole (DAPI) (blue) demonstrate GFP expression in regenerating fibers. Upper row images were taken using 10× objective lens, lower row with 20× objective. Scale bars = 100 μm.

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